Impact of the different preparation methods to obtain human adipose-derived stromal vascular fraction cells (AD-SVFs) and human adipose-derived mesenchymal stem cells (AD-MSCs): Enzymatic digestion versus mechanical centrifugation

Autologous therapies using adipose-derived stromal vascular fraction (AD-SVFs) and adult adipose-derived mesenchymal stem cells (AD-MSCs) warrant careful preparation of the harvested adipose tissue. Currently, no standardized technique for this preparation exists. Processing quantitative standards (PQSs) define manufacturing quantitative variables (such as time, volume, and pressure). Processing qualitative standards (PQLSs) define the quality of the materials and methods in manufacturing. The purpose of the review was to use PQSs and PQLSs to report the in vivo and in vitro results obtained by different processing kits that use different procedures (enzymatic vs. non-enzymatic) to isolate human AD-SVFs/AD-MSCs. PQSs included the volume of fat tissue harvested and reagents used, the time/gravity of centrifugation, and the time, temperature, and tilt level/speed of incubation and/or centrifugation. PQLSs included the use of a collagenase, a processing time of 30 min, kit weight, transparency of the kit components, the maintenance of a closed sterile processing environment, and the use of a small centrifuge and incubating rocker. Using a kit with the PQSs and PQLSs described in this study enables the isolation of AD-MSCs that meet the consensus quality criteria. As the discovery of new critical quality attributes (CQAs) of AD-MSCs evolve with respect to purity and potency, adjustments to these benchmark PQSs and PQLs will hopefully isolate AD-MSCs of various CQAs with greater reproducibility, quality, and safety. Confirmatory studies will no doubt need to be completed

Toll-like Receptor 4 Expression, Interleukin-6, -8 and Ccl-20 Release, and NF-KB Translocation in Human Periodontal Ligament Mesenchymal Stem Cells Stimulated with LPS-P. Gingivalis

Periodontal diseases, the major public health problem of the oral cavity, are clinically characterized by inflammation of the periodontal connective tissue that ultimately induces the destruction of periodontal tissue and the loss of alveolar bone. In chronic periodontitis, as well as aggressive periodontitis, the anaerobic gram-negative bacterium Porphyromonas gingivalis (P. gingivalis) is implicated. The pathogenicity of P. gingivalis is exerted by a wide variety of factors, including lipopolysaccharides (LPSs). LPSs activate the innate immune response during Gram-negative bacterial infections through the Toll-like receptor 4 (TLR-4)/myeloid differentiation protein 2 (MD-2) complex. In this study, the expression of TLR-4, the cell growth, the cytokine release, and the nuclear factor-KB (NF-kB) transcription factor expression in response to LPS- P.Gingivalis (LPS-G) were examined in Human Periodontal Ligament Mesenchymal Stem Cells (PDL-MSCs). The results obtained demonstrate that, in basal conditions, human PDL-MSCs express high levels of TLR-4. In inflammatory conditions mimicked by LPS-G challenge, the MTT assay carried out at different treatment times demonstrated the decrease of the cell growth. Moreover, the recognition of P. gingivalis components by TLR-4 culminated with the activation of secretion of inflammatory mediators such as: IL-6, IL-8 and CCL-20, and with the up-regulation of NF-kB, which was translocated into the nucleus. Our data intended to specify that TLR-4 expressed by PDL-MSCs is functional and plays a key role in inflammation

Inhibition of LPS-Induced Inflammatory Response of Oral Mesenchymal Stem Cells in the Presence of Galectin-3

Galectin-3 (GAL-3) is a beta-galactoside binding lectin produced by mesenchymal stem cells (MSCs) and other cell sources under inflammatory conditions. Several studies have reported that GAL-3 exerts an anti-inflammatory action, regulated by its natural ligand GAL-3 BP. In the present study, we aimed to assess the GAL-3 mediated regulation of the MSC function in an LPS-induced inflammation setting. Human gingival mesenchymal stem cells (hGMSCs) were stimulated in vitro with LPSs; the expression of TLR4, NFκB p65, MyD88 and NALP3 were assessed in the hGMSCs via immunofluorescence imaging using confocal microscopy, Western blot assay, and RT-PCR before and after the addition of GAL-3, both alone and with the addition of its inhibitors. LPSs stimulated the expression of TLR4, NFκB p65, MyD88 and NALP3 in hGMSCs, which was inhibited by GAL-3. The addition of either GAL3-BP or the antibody to GAL-3 were able to revert the GAL-3-mediated effects, restoring the expression of TLR4, NFκB p65, MyD88 and NALP3. GAL-3 induces the downregulation of the LPS-induced inflammatory program in MSCs

Impact of the different preparation methods to obtain autologous non\u2010activated platelet\u2010rich plasma (A\u2010PRP) and activated platelet\u2010rich plasma (AA\u2010PRP) in plastic surgery: Wound healing and hair regrowth evaluation

Autologous therapies using platelet\u2010rich plasma (PRP) need meticulous preparation\u2014currently, no standardised preparation technique exists. Processing Quantitative Standards (PQSs) define manufacturing quantitative variables (such as time, volume and pressure). Processing Qualitative Standards (PQLSs) define the quality of the materials and methods of manufacturing. The aim of this review is to use existing PQSs and PQLs to report the in vivo/in vitro results obtained by using different Kits, that utilise different procedures (classified as Closed\u2010Technique and Opened\u2010Technique) to isolate autologous human activated (AA\u2010PRP) or non\u2010activated PRP (A\u2010PRP). PQSs included the volumes of blood collected as well as the reagents used, the time/gravity of centrifugation, and the duration, temperature and tilt level/speed of centrifugation. PQLSs included the use of Calcium Chloride CaCl2, Kit weight, transparency of Kit components, the maintenance of a closed sterile processing environment and the use of a small centrifuge. Eight CE marked devices for PRP extraction were evaluated: Angel\uae, Biomed\uae, Cascade\uae and Selphyl\uae, Mag\u201018\uae, i\u2010Stem\uae, MyCells\uae and Regenlab\uae. Using a Kit with the PQSs and PQLSs described in this study enables the isolation of A\u2010PRP, thereby meeting consensus quality criteria. As our understanding of Critical Quality Attributes (CQAs) of A\u2010PRP continues to evolve, especially with respect to purity and potency, adjustments to these benchmark PQSs and PQLs will hopefully help isolate A\u2010PRP of desired CQAs with greater reproducibility, quality, and safety. Confirmatory studies will no doubt need to be completed

A new class of glycomimetic drugs to prevent free fatty acid-induced endothelial dysfunction

Background: Carbohydrates play a major role in cell signaling in many biological processes. We have developed a set of glycomimetic drugs that mimic the structure of carbohydrates and represent a novel source of therapeutics for endothelial dysfunction, a key initiating factor in cardiovascular complications. Purpose: Our objective was to determine the protective effects of small molecule glycomimetics against free fatty acid­induced endothelial dysfunction, focusing on nitric oxide (NO) and oxidative stress pathways. Methods: Four glycomimetics were synthesized by the stepwise transformation of 2,5­dihydroxybenzoic acid to a range of 2,5­substituted benzoic acid derivatives, incorporating the key sulfate groups to mimic the interactions of heparan sulfate. Endothelial function was assessed using acetylcholine­induced, endotheliumdependent relaxation in mouse thoracic aortic rings using wire myography. Human umbilical vein endothelial cell (HUVEC) behavior was evaluated in the presence or absence of the free fatty acid, palmitate, with or without glycomimetics (1µM). DAF­2 and H2DCF­DA assays were used to determine nitric oxide (NO) and reactive oxygen species (ROS) production, respectively. Lipid peroxidation colorimetric and antioxidant enzyme activity assays were also carried out. RT­PCR and western blotting were utilized to measure Akt, eNOS, Nrf­2, NQO­1 and HO­1 expression. Results: Ex vivo endothelium­dependent relaxation was significantly improved by the glycomimetics under palmitate­induced oxidative stress. In vitro studies showed that the glycomimetics protected HUVECs against the palmitate­induced oxidative stress and enhanced NO production. We demonstrate that the protective effects of pre­incubation with glycomimetics occurred via upregulation of Akt/eNOS signaling, activation of the Nrf2/ARE pathway, and suppression of ROS­induced lipid peroxidation. Conclusion: We have developed a novel set of small molecule glycomimetics that protect against free fatty acidinduced endothelial dysfunction and thus, represent a new category of therapeutic drugs to target endothelial damage, the first line of defense against cardiovascular disease

Optical coherence tomography accurately identifies intermediate atherosclerotic lesions--an in vivo evaluation in the rabbit carotid artery

Abstract Objective: We tested the ability of optical coherence tomography (OCT) to identify very early stages of atherosclerosis in vivo. Methods: Twelve New Zealand white male rabbits (weight 3.5–4.0 kg) underwent perivascular electrical injury of the common carotid arteries, and were then fed a cholesterol-rich diet. At 43±16 (range 27–63) days after injury, arteries were imaged by OCT, then rabbits were euthanized and vessels processed for histology. Results: A total of 14 carotid arteries were imaged by OCT and histology; 22 atherosclerotic lesions were identified, 16 (73%) occurring at the site of the electrical injury. At histology, 4 lesions were defined as Stary type I (isolated macrophages), 8 as type II (intracellular lipid accumulations), and 10 as type III (small extracellular lipid pools). No advanced (≥type IV) lesions were documented. OCT failed to detect any type I lesions, but correctly defined a minority (2/8, 25%) of type II lesions and the majority (8/10, 80%) of type III lesions. For type III lesions, sensitivity, specificity and diagnostic accuracy of OCT were 80%, 95%, and 95%, respectively. Conclusions: OCT can accurately detect intermediate (type-III) atherosclerotic lesions in vivo, but still fails to identify earlier stages of atherosclerosis

Does optical coherence tomography identify arterial healing after stenting? An in vivo comparison with histology, in a rabbit carotid model

ABSTRACT Objective: To verify whether optical coherence tomography (OCT) can accurately monitor the occurrence of arterial healing after stenting. Setting: Delayed stent endothelialisation may predispose to stent thrombosis. OCT is a high-resolution intravascular imaging technique that accurately identifies stent struts and arterial tissues. Design and interventions: Eight New Zealand white rabbits underwent the implantation of single bare metal stents (diameter 2–2.5 mm, length 8–13 mm) in the right common carotid artery through the external carotid artery. After a median of 11 days (range 2–28), the stented arteries were visualised by OCT, with images acquired at a pull-back speed of 0.5 mm/sec. The rabbits were then euthanised, vessels were formalin-fixed and finally processed for histopathology. Results: We analysed 32 cross-sections from eight stented carotid arteries, for a total of 384 stent struts. OCT detected all of the stent struts in 30 of 32 crosssections (93.7%), and correctly identified the presence/ absence of tissue for every strut. Histological and OCT measurements of mean neointima thickness (0.135 (SD 0.079) mm and 0.145 (SD 0.085) mm, respectively, p=NS) were similar and closely related (r=0.85, p,0.001). Neointima area progressively increased with longer time intervals from stent deployment to sacrifice; histological and OCT measurements were similar for each time interval. The intra-observer and interobserver reproducibility of OCT neointima measurements were excellent (R2=0.90 and 0.88, respectively). Conclusions: OCT is a promising means for monitoring stent strut coverage and vessel wall healing in vivo, the relevance of which will become even more significant with the increasing use of drug-eluting stents